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Developmental Cell Apr 2024Dynamic changes in three-dimensional cell shape are important for tissue form and function. In the developing Drosophila eye, photoreceptor differentiation requires the...
Dynamic changes in three-dimensional cell shape are important for tissue form and function. In the developing Drosophila eye, photoreceptor differentiation requires the progression across the tissue of an epithelial fold known as the morphogenetic furrow. Morphogenetic furrow progression involves apical cell constriction and movement of apical cell edges. Here, we show that cells progressing through the morphogenetic furrow move their basal edges in opposite direction to their apical edges, resulting in a cellular tilting movement. We further demonstrate that cells generate, at their basal side, oriented, force-generating protrusions. Knockdown of the protein kinase Src42A or photoactivation of a dominant-negative form of the small GTPase Rac1 reduces protrusion formation. Impaired protrusion formation stalls basal cell movement and slows down morphogenetic furrow progression and photoreceptor differentiation. This work identifies a cellular tilting mechanism important for the generation of dynamic tissue shape changes and cell differentiation.
PubMed: 38692272
DOI: 10.1016/j.devcel.2024.04.011 -
Journal of Medicinal Chemistry May 2024Retinitis pigmentosa (RP) is a form of retinal degeneration affecting a young population with an unmet medical need. Photoreceptor degeneration has been associated with...
Retinitis pigmentosa (RP) is a form of retinal degeneration affecting a young population with an unmet medical need. Photoreceptor degeneration has been associated with increased guanosine 3',5'-cyclic monophosphate (cGMP), which reaches toxic levels for photoreceptors. Therefore, inhibitory cGMP analogues attract interest for RP treatments. Here we present the synthesis of dithio-CN03, a phosphorodithioate analogue of cGMP, prepared using the H-phosphonothioate route. Two crystal modifications were identified as a trihydrate and a tetrahydrofuran monosolvates. Dithio-CN03 featured a lower aqueous solubility than its -phosphorothioate counterpart CN03, a drug candidate, and this characteristic might be favorable for sustained-release formulations aimed at retinal delivery. Dithio-CN03 was tested in vitro for its neuroprotective effects in photoreceptor models of RP. The comparison of dithio-CN03 to CN03 and its diastereomer -CN03, and to their phosphate derivative oxo-CN03 identifies dithio-CN03 as the compound with the highest efficacy in neuroprotection and thus as a promising new candidate for the treatment of RP.
Topics: Cyclic GMP; Cyclic Nucleotide Phosphodiesterases, Type 6; Guanosine Monophosphate; Neuroprotective Agents; Retinal Degeneration; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Structure-Activity Relationship
PubMed: 38688030
DOI: 10.1021/acs.jmedchem.4c00586 -
Frontiers in Neuroscience 2024In the eye, cells from the retinal pigment epithelium (RPE) facing the neurosensory retina exert several functions that are all crucial for long-term survival of...
In the eye, cells from the retinal pigment epithelium (RPE) facing the neurosensory retina exert several functions that are all crucial for long-term survival of photoreceptors (PRs) and vision. Among those, RPE cells phagocytose under a circadian rhythm photoreceptor outer segment (POS) tips that are constantly subjected to light rays and oxidative attacks. The MerTK tyrosine kinase receptor is a key element of this phagocytic machinery required for POS internalization. Recently, we showed that MerTK is subjected to the cleavage of its extracellular domain to finely control its function. In addition, monocytes in retinal blood vessels can migrate inside the inner retina and differentiate into macrophages expressing MerTK, but their role in this context has not been studied yet. We thus investigated the ocular phenotype of MerTK cleavage-resistant (MerTK) mice to understand the relevance of this characteristic on retinal homeostasis at the RPE and macrophage levels. MerTK retinae appear to develop and function normally, as observed in retinal sections, by electroretinogram recordings and optokinetic behavioral tests. Monitoring of MerTK and control mice between the ages of 3 and 18 months showed the development of large degenerative areas in the central retina as early as 4 months when followed monthly by optical coherence tomography (OCT) plus fundus photography (FP)/autofluorescence (AF) detection but not by OCT alone. The degenerative areas were associated with AF, which seems to be due to infiltrated macrophages, as observed by OCT and histology. MerTK RPE primary cultures phagocytosed less POS , while , the circadian rhythm of POS phagocytosis was deregulated. Mitochondrial function and energy production were reduced in freshly dissected RPE/choroid tissues at all ages, thus showing a metabolic impairment not present in macrophages. RPE anomalies were detected by electron microscopy, including phagosomes retained in the apical area and vacuoles. Altogether, this new mouse model displays a novel phenotype that could prove useful to understanding the interplay between RPE and PRs in inflammatory retinal degenerations and highlights new roles for MerTK in the regulation of the energetic metabolism and the maintenance of the immune privilege in the retina.
PubMed: 38680449
DOI: 10.3389/fnins.2024.1256522 -
Cell Reports May 2024Cellular retinaldehyde-binding protein (CRALBP) supports production of 11-cis-retinaldehyde and its delivery to photoreceptors. It is found in the retinal pigment...
Cellular retinaldehyde-binding protein (CRALBP) supports production of 11-cis-retinaldehyde and its delivery to photoreceptors. It is found in the retinal pigment epithelium (RPE) and Müller glia (MG), but the relative functional importance of these two cellular pools is debated. Here, we report RPE- and MG-specific CRALBP knockout (KO) mice and examine their photoreceptor and visual cycle function. Bulk visual chromophore regeneration in RPE-KO mice is 15-fold slower than in controls, accounting for their delayed rod dark adaptation and protection against retinal phototoxicity, whereas MG-KO mice have normal bulk visual chromophore regeneration and retinal light damage susceptibility. Cone pigment regeneration is significantly impaired in RPE-KO mice but mildly affected in MG-KO mice, disclosing an unexpectedly strong reliance of cone photoreceptors on the RPE-based visual cycle. These data reveal a dominant role for RPE-CRALBP in supporting rod and cone function and highlight the importance of RPE cell targeting for CRALBP gene therapies.
Topics: Animals; Mice; Carrier Proteins; Ependymoglial Cells; Mice, Inbred C57BL; Mice, Knockout; Retinal Cone Photoreceptor Cells; Retinal Pigment Epithelium; Retinal Pigments; Retinal Rod Photoreceptor Cells; Male; Female
PubMed: 38676924
DOI: 10.1016/j.celrep.2024.114143 -
International Journal of Molecular... Apr 2024In this review, we outline our current understanding of the mechanisms involved in the absorption, storage, and transport of dietary vitamin A to the eye, and the... (Review)
Review
In this review, we outline our current understanding of the mechanisms involved in the absorption, storage, and transport of dietary vitamin A to the eye, and the trafficking of rhodopsin protein to the photoreceptor outer segments, which encompasses the logistical backbone required for photoreceptor cell function. Two key mechanisms of this process are emphasized in this manuscript: ocular and systemic vitamin A membrane transporters, and rhodopsin transporters. Understanding the complementary mechanisms responsible for the generation and proper transport of the retinylidene protein to the photoreceptor outer segment will eventually shed light on the importance of genes encoded by these proteins, and their relationship on normal visual function and in the pathophysiology of retinal degenerative diseases.
Topics: Rhodopsin; Humans; Vitamin A; Animals; Photoreceptor Cells, Vertebrate; Photoreceptor Cells; Biological Transport
PubMed: 38673863
DOI: 10.3390/ijms25084278 -
Developmental Cell Apr 2024Proteotoxic stress drives numerous degenerative diseases. Cells initially adapt to misfolded proteins by activating the unfolded protein response (UPR), including...
Proteotoxic stress drives numerous degenerative diseases. Cells initially adapt to misfolded proteins by activating the unfolded protein response (UPR), including endoplasmic-reticulum-associated protein degradation (ERAD). However, persistent stress triggers apoptosis. Enhancing ERAD is a promising therapeutic approach for protein misfolding diseases. The ER-localized Zn transporter ZIP7 is conserved from plants to humans and required for intestinal self-renewal, Notch signaling, cell motility, and survival. However, a unifying mechanism underlying these diverse phenotypes was unknown. In studying Drosophila border cell migration, we discovered that ZIP7-mediated Zn transport enhances the obligatory deubiquitination of proteins by the Rpn11 Zn metalloproteinase in the proteasome lid. In human cells, ZIP7 and Zn are limiting for deubiquitination. In a Drosophila model of neurodegeneration caused by misfolded rhodopsin (Rh1), ZIP7 overexpression degrades misfolded Rh1 and rescues photoreceptor viability and fly vision. Thus, ZIP7-mediated Zn transport is a previously unknown, rate-limiting step for ERAD in vivo with therapeutic potential in protein misfolding diseases.
PubMed: 38670102
DOI: 10.1016/j.devcel.2024.04.003 -
Journal of Controlled Release :... Jun 2024Remodeling retinal Müller glial fate, including gliosis inhibition and pro-reprogramming, represents a crucial avenue for treating degenerative retinal diseases. Stem...
Remodeling retinal Müller glial fate, including gliosis inhibition and pro-reprogramming, represents a crucial avenue for treating degenerative retinal diseases. Stem cell transplantation exerts effects on modulating retinal Müller glial fate. However, the optimized stem cell products and the underlying therapeutic mechanisms need to be investigated. In the present study, we found that retinal progenitor cells from human embryonic stem cell-derived retinal organoids (hERO-RPCs) transferred extracellular vesicles (EVs) into Müller cells following subretinal transplantation into RCS rats. Small EVs from hERO-RPCs (hERO-RPC-sEVs) were collected and were found to delay photoreceptor degeneration and protect retinal function in RCS rats. hERO-RPC-sEVs were taken up by Müller cells both in vivo and in vitro, and inhibited gliosis while promoting early dedifferentiation of Müller cells. We further explored the miRNA profiles of hERO-RPC-sEVs, which suggested a functional signature associated with neuroprotection and development, as well as the regulation of stem cell and glial fate. Mechanistically, hERO-RPC-sEVs might regulate the fate of Müller cells by miRNA-mediated nuclear factor I transcription factors B (NFIB) downregulation. Collectively, our findings offer novel mechanistic insights into stem cell therapy and promote the development of EV-centered therapeutic strategies.
Topics: Extracellular Vesicles; Animals; MicroRNAs; Humans; Retinal Degeneration; Ependymoglial Cells; Organoids; Rats; Retina; Human Embryonic Stem Cells; Stem Cell Transplantation; Gliosis; Cell Differentiation; Stem Cells
PubMed: 38663753
DOI: 10.1016/j.jconrel.2024.04.036 -
ELife Apr 2024Retinitis pigmentosa (RP), a heterogenous group of inherited retinal disorder, causes slow progressive vision loss with no effective treatments available. Mutations in...
Retinitis pigmentosa (RP), a heterogenous group of inherited retinal disorder, causes slow progressive vision loss with no effective treatments available. Mutations in the rhodopsin gene () account for ~25% cases of autosomal dominant RP (adRP). In this study, we describe the disease characteristics of the first-ever reported mono-allelic copy number variation (CNV) in as a novel cause of adRP. We (a) show advanced retinal degeneration in a male patient (68 years of age) harboring four transcriptionally active intact copies of rhodopsin, (b) recapitulated the clinical phenotypes using retinal organoids, and (c) assessed the utilization of a small molecule, Photoregulin3 (PR3), as a clinically viable strategy to target and modify disease progression in RP patients associated with -CNV. Patient retinal organoids showed photoreceptors dysgenesis, with rod photoreceptors displaying stunted outer segments with occasional elongated cilia-like projections (microscopy); increased mRNA expression (quantitative real-time PCR [qRT-PCR] and bulk RNA sequencing); and elevated levels and mislocalization of rhodopsin protein (RHO) within the cell body of rod photoreceptors (western blotting and immunohistochemistry) over the extended (300 days) culture time period when compared against control organoids. Lastly, we utilized PR3 to target , an upstream regulator of , to alter expression and observed a partial rescue of RHO protein localization from the cell body to the inner/outer segments of rod photoreceptors in patient organoids. These results provide a proof-of-principle for personalized medicine and suggest that expression requires precise control. Taken together, this study supports the clinical data indicating that RHO-CNV associated adRPdevelops as a result of protein overexpression, thereby overloading the photoreceptor post-translational modification machinery.
Topics: Aged; Humans; Male; DNA Copy Number Variations; Organoids; Retinitis Pigmentosa; Rhodopsin
PubMed: 38661530
DOI: 10.7554/eLife.90575 -
BioRxiv : the Preprint Server For... Apr 2024Age is the principal risk factor for neurodegeneration in both the retina and brain. The retina and brain share many biological properties; thus, insights into retinal...
BACKGROUND
Age is the principal risk factor for neurodegeneration in both the retina and brain. The retina and brain share many biological properties; thus, insights into retinal aging and degeneration may shed light onto similar processes in the brain. Genetic makeup strongly influences susceptibility to age-related retinal disease. However, studies investigating retinal aging have not sufficiently accounted for genetic diversity. Therefore, examining molecular aging in the retina across different genetic backgrounds will enhance our understanding of human-relevant aging and degeneration in both the retina and brain-potentially improving therapeutic approaches to these debilitating conditions.
METHODS
Transcriptomics and proteomics were employed to elucidate retinal aging signatures in nine genetically diverse mouse strains (C57BL/6J, 129S1/SvlmJ, NZO/HlLtJ, WSB/EiJ, CAST/EiJ, PWK/PhK, NOD/ShiLtJ, A/J, and BALB/cJ) across lifespan. These data predicted human disease-relevant changes in WSB and NZO strains. Accordingly, B6, WSB and NZO mice were subjected to human-relevant examinations at 4, 8, 12, and/or 18M, including: slit lamp, fundus imaging, optical coherence tomography, fluorescein angiography, and pattern/full-field electroretinography. Retinal morphology, vascular structure, and cell counts were assessed
RESULTS
We identified common molecular aging signatures across the nine mouse strains, which included genes associated with photoreceptor function and immune activation. Genetic background strongly modulated these aging signatures. Analysis of cell type-specific marker genes predicted age-related loss of photoreceptors and retinal ganglion cells (RGCs) in WSB and NZO, respectively. Fundus exams revealed retinitis pigmentosa-relevant pigmentary abnormalities in WSB retinas and diabetic retinopathy (DR)-relevant cotton wool spots and exudates in NZO retinas. Profound photoreceptor dysfunction and loss were confirmed in WSB. Molecular analyses indicated changes in photoreceptor-specific proteins prior to loss, suggesting photoreceptor-intrinsic dysfunction in WSB. In addition, age-associated RGC dysfunction, loss, and concomitant microvascular dysfunction was observed in NZO mice. Proteomic analyses revealed an early reduction in protective antioxidant processes, which may underlie increased susceptibility to DR-relevant pathology in NZO.
CONCLUSIONS
Genetic context is a strong determinant of retinal aging, and our multi-omics resource can aid in understanding age-related diseases of the eye and brain. Our investigations identified and validated WSB and NZO mice as improved preclinical models relevant to common retinal neurodegenerative diseases.
PubMed: 38659747
DOI: 10.1101/2024.04.16.589625 -
The Journal of Neuroscience : the... Jun 2024Hexanucleotide repeat expansions within the gene are the most common cause of the neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and frontotemporal...
Hexanucleotide repeat expansions within the gene are the most common cause of the neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). This disease-causing expansion leads to a reduction in C9ORF72 expression levels in patients, suggesting loss of C9ORF72 function could contribute to disease. To further understand the consequences of C9ORF72 deficiency in vivo, we generated a mutant zebrafish line. Analysis of the adult female spinal cords revealed no appreciable neurodegenerative pathology such as loss of motor neurons or increased levels of neuroinflammation. However, detailed examination of adult female retinas showed prominent neurodegenerative features, including a decrease in retinal thickness, gliosis, and an overall reduction in neurons of all subtypes. Analysis of rod and cone cells within the photoreceptor layer showed a disturbance in their outer segment structure and rhodopsin mislocalization from rod outer segments to their cell bodies and synaptic terminals. Thus, C9ORF72 may play a previously unappreciated role in retinal homeostasis and suggests C9ORF72 deficiency can induce tissue specific neuronal loss.
Topics: Animals; Zebrafish; Female; C9orf72 Protein; Retina; Animals, Genetically Modified; Zebrafish Proteins; Proteins; Retinal Degeneration; Neurodegenerative Diseases; Spinal Cord
PubMed: 38658168
DOI: 10.1523/JNEUROSCI.2128-23.2024